A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in Actinobacteria

ABSTRACT Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3′,5′-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology. IMPORTANCE Since the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

The maintenance and stability of c-di-AMP are fundamental to c-di-AMP-producing species.Elevated c-di-AMP levels result in aberrant physiology, given the expanding number of recognized organisms that synthesize c-di-AMP and the importance it plays in the diverse environments that bacteria encounter (18).Similar to other second messengers, c-di-AMP levels must be controlled to prevent toxic accumulation.These depletion strategies appear to be regulated by environmental and intracellular signals and are integrated with other stress response pathways.Upon environmental stimula tion, changes in intracellular c-di-AMP levels rely on the activity of diadenylate cyclase (DAC) domain-containing proteins or c-di-AMP-specific phosphodiesterase (10).The DAC domain was first identified in the DNA integrity scanning protein (DisA) (19).To date, four classes of DACs have been characterized-DisA, CdaA, CdaS, and CdaM-while most organisms contain only one type of DAC (3).
Despite significant progress in our understanding of c-di-AMP synthesis and hydrolysis, the signals that adjust the intracellular c-di-AMP concentration and how this information is converted into feedback signaling, as well as the regulatory mechanism behind its synthesis and degradation, remain largely unknown.In bacteria, DAC domain proteins are most frequently found in the Gram-positive Firmicutes and within the phylum Actinobacteria (2).Actinobacteria are ubiquitous, primarily soil-dwelling bacteria with a complicated developmental life cycle involving the progression from vegetative growth to the production of reproductive aerial hyphae that differentiate into chains of exospores (20).Entry into development coincides with the biosynthesis of numerous secondary metabolites that serve as the most abundant source of clinically important antibiotics and provide other medically important drugs, for instance, anti-cancer agents and immunosuppressants (21)(22)(23).Consequently, there is considerable interest in understanding the mechanism that relate to this developmental transition.
Recent work on protein acetylation in Gram-positive Actinobacteria has highligh ted the importance of acetylation as a signal that provides functional diversity and regulation by modifying proteins to respond to diverse stimuli, which provides a direct link between cell metabolism and signal transduction, transcriptional regulation, cell growth, and pathogenicity (24,25).Acetylation is one of many posttranslational modifications (PTMs) that are important in biological systems (26).Bacterial acetylation depends on both enzymatic and nonenzymatic mechanisms of acetylation, and both mechanisms can control the properties of that protein, such as enzymatic activity, localization, stability, or interactions with other molecules (27,28).
Here, we show that c-di-AMP maintenance is processed by a network of acetyl phosphate (AcP)-induced acetylation.Acetylation induced by AcP disrupts the multime rization of its substrates in Saccharopolyspora erythraea, leading to impaired c-di-AMP synthesis via K66 acetylation of DisA.Conversely, acetylation of DasR at K78 relieves the transcriptional inhibition of disA and promotes intracellular c-di-AMP levels, altogether supporting c-di-AMP homeostasis.Altered c-di-AMP levels either through DisA or DasR acetylation could cause a metabolic imbalance, leading to an alert signal for develop mental transitions.These findings highlight the crucial role of AcP in balancing c-di-AMP levels and suggest that this unique regulation is likely a necessary feature in Actinobacte ria.

AcP-dependent acetylation directly impairs the DAC activity of DisA
Our previous study revealed that nitrogen starvation leads to AcP accumulation in S. erythraea, thus exerting a general effect on the global acetylation level in an AcPmediated manner (24).The S. erythraea genome encodes only one type of DAC (DisA, SACE_0435) (Kyoto Encyclopedia of Genes and Genomes database, http://www.kegg.jp/).To explore the role of AcP in intracellular c-di-AMP homeostasis, we first tested whether DisA is a substrate of acetylation in Gram-positive S. erythraea.The acetylation status of DisA in vivo was tested by immunoprecipitation (IP) and immunoblotting (IB) analyses.DisA from S. erythraea cells was immunoprecipitated with an antibody against DisA.Acetyl-lysine levels were detected in DisA immunoprecipitates with an anti-AcK antibody.As shown in Fig. 1A, DisA was hyperacetylated under nitrogen starvation conditions.In vitro acetylation also showed that DisA could be acetylated by AcP (Fig. 1B).These results demonstrated that the DisA protein is a new acetylation substrate both in vitro and in vivo.To investigate the effect of acetylation on the DAC activity of DisA, DisA was incubated with or without AcP for 5 h.The activities of nonacetylated DisA (DisA WT ) and acetylated DisA (DisA AcP ) enzymes were determined.DisA AcP activity was reduced by ∼75%, indicating that AcP-dependent acetylation effectively decreased its DAC activity (Fig. 1C).DisA forms a large octamer that possesses diadenylate cyclase activity (19).The secondary structure of DisA was then examined by circular dichroism (CD) assay.The far-UV CD spectra showed that AcP-dependent acetylation caused an influence on the DisA structure with lower α-helicity and a concomitant increase in anti-parallel and random coil structures compared with DisA WT (Fig. 1D; Table S1).These results suggested that AcP-dependent acetylation likely plays a key role in the synthesis of c-di-AMP.

DisA is inactive mainly upon acetylation of K66
To determine the acetylation sites of DisA, the in vitro and in vivo acetylated DisA proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/ MS).As listed in Table S2 and Fig. S1 through S3, two acetylated peptides containing K66 and K284 were identified in AcP-dependent acetylated DisA, while only K66 was identified in endogenous DisA.K66 is located in the DAC domain, which mediates diadenylate cyclase activity (Fig. 1E).To examine the K66 acetylation effect in detail, we introduced substitutions at the acetylated site K66 to generate the variants DisA K66Q  and DisA K66R based on the principle that glutamine (Q) can serve as a structural mimic for acetyl-lysine and that arginine (R) serves as a genetic mimic of unacetylated lysine (29).The DisA WT , DisA AcP , DisA K66Q , and DisA K66R proteins were then subjected to both chemical cross-linking and DAC assays, and we found that DisA K66R showed equivalent or similar intensities of cross-linking bands comparable to those obtained for DisA WT (Fig. 1F).However, the DisA K66Q mutant displayed a significantly reduced octamer form and an increased monomer form (Fig. 1F).These results revealed that the K66 residue was critical for DisA octamerization.The results of the DAC assay (Fig. 1G) led to the same conclusion that DisA K66Q exhibited weak DAC activity.These results indicated that AcP-dependent acetylation at K66 dominated the impairment of DisA DAC activity.
Bacterial c-di-AMP synthesis is catalyzed by the DAC activity of DisA.To investigate whether K66 acetylation affects c-di-AMP synthesis in S. erythraea, we constructed DisA and its mutant overexpression strains as previously described (30).With the wild-type (WT) strain used as a control, we observed that both OdisA WT and OdisA K66R had relatively longer lag periods than the other strains (Fig. 2A); a possible reason might be that the overexpression of active DisA affects bacterial growth.The constructed strains were then analyzed by real-time RT-PCR, and the three DisA-overexpressing strains showed comparable disA transcription levels, which were more than a 15-fold greater than those of the WT strain (Fig. 2B).We next examined the effect of DisA and its mutant overexpres sion on intracellular c-di-AMP levels.A more than 75% drop in the intracellular c-di-AMP level of the OdisA K66Q strain was observed in comparison to that of the OdisA WT strain, and the OdisA K66R strain performed like the OdisA WT strain (Fig. 2C).In functional terms, these findings supported that DisA inactivation resulting from K66 acetylation directly abrogated its DAC activity and hence intracellular c-di-AMP synthesis directly.

AcP-dependent acetylation eliminates the transcriptional control of disA via DasR deactivation
Of note, disA was recently identified as a target gene of the global regulator DasR according to our latest study (31), in which DasR exerts direct transcriptional repres sion of c-di-AMP synthesis.We reasoned that AcP could also have an effect on the transcriptional control of disA expression via DasR acetylation.To test our hypothesis, the acetylation status of the DasR immunoprecipitates was determined with an anti-AcK antibody.As shown in Fig. 3A, DasR was hyperacetylated under nitrogen starvation conditions.In vitro acetylation further confirmed that DasR was acetylated by AcP (Fig. 3B).To investigate the effect of acetylation on the DNA-binding activity of the DasR regulator, electrophoretic mobility shift assay (EMSA) was performed using nonacety lated DasR (DasR WT ) and AcP-acetylated DasR (DasR AcP ).As shown in Fig. 3C, DasR AcP showed weak DNA binding, as evidenced by the faint mobility shift.DasR belongs to the GntR-family regulators and interacts with DNA as a dimer (32).To determine whether AcP-induced acetylation influences DasR dimerization, a CD assay was performed.The Far-ultraviolet circular dichroism (Far-UV CD) spectra showed an increase in the α-helical content and a concomitant decrease in parallel and random coil structures compared with those of DasR WT , suggesting that acetylation altered the secondary structure of DasR (Fig. 3D; Table S1).
The proteomic data further showed that K78 was the only acetylation site identified in endogenous DasR (Table S2; Fig. S4 through 8).K78 is located in the GntR HTH domain, indicating a potential role of DNA binding (Fig. 3E).To verify this, we introduced substitutions at K78 to generate the DasR K78Q and DasR K78R variants.Together with DasR WT , the three proteins were subjected to EMSA, and we found that DasR K78R performed similarly to DasR WT , while DasR K78Q showed weak DNA binding, as evidenced by the slight mobility shift (Fig. 3F).To confirm this, we measured the Kd between DasR or its mutants and DNA using a biolayer interferometry (BLI) assay.As shown in Fig. 3G, DasR-DNA had a binding affinity of Kd ∼0.4 µM, whereas the Kd was ∼20 µM, a 50-fold increase for DasR K78Q -DNA, which was consistent with the data shown in Fig. 3E and F. Additionally, we found a reduced dimer form and an increased monomer form in DasR K78Q (Fig. 3H), indicating that the dimerization function was also affected.These results reflected the inability of DasR to efficiently bind to the disA promoter after AcP-dependent acetylation at K78.

DasR acetylation at K78 stimulates disa transcription and c-di-AMP synthesis
To confirm and extend K78 acetylation of DasR in cells, we constructed S. erythraea strains overexpressing DasR WT and DasR K78Q using the E. coli-S.erythraea integrative shuttle vector pIB139 (33).The corresponding strains were subjected to chromatin immunoprecipitation sequencing (ChIP-seq) experiments using a specific anti-DasR antibody.The total input DNA (nonimmunoprecipitated) from each strain was also subjected to sequencing.Both the DasR WT and DasR K78Q signals were widely distributed at transcription start sites, with a sharp single peak (Fig. 4A).The total detected peaks showed downregulated binding signals in the DasR K78Q strain compared with the DasR WT strain (Fig. 4B).Consistently, representative results from the visualization and verification of disA during exposure to DasR K78Q overexpression illustrated that the ChIP-seq peak changed at the individual gene level (Fig. 4C; Fig. S9).We therefore concluded that K78 acetylation attenuated the global outcomes of DasR binding to its targets, including disA.DasR, which predominantly functions as a global repressor, has a negative effect on disA expression (31).We then confirmed the ChIP-seq data by real-time RT-PCR to analyze the transcription of disA.As expected, the repression of disA was released in DasR K78Q (Fig. 4D).In addition, the intracellular c-di-AMP concentration showed a consistent trend (Fig. 4E), supporting the idea that AcP signals through DasR to control the intracellular c-di-AMP level.

c-di-AMP homeostasis mediates multicellular differentiation
The above two lines of evidence through transcriptional regulation and posttranslational regulation outline that AcP is a pivotal modulator of DisA, thereby providing a mecha nism of c-di-AMP homeostasis control.First, DasR activation is hampered during acetylation at K78, indicating that this strain experiences excess c-di-AMP resulting from AcP-mediated indirect regulation.Second, DisA acetylation at K66 led to lower intracellu lar c-di-AMP amounts, indicating direct regulation caused by AcP-mediated acetylation.As cellular c-di-GMP levels were shown to control developmental transition in filamentous actinobacteria (20,34,35), we speculated that c-di-AMP might confer a similar function.To explore the possibility of this phenomenon being involved in developmental physiology and whether it is associated with AcP-mediated acetylation, the S. erythraea WT, OdisA K66Q , OdisA K66R , OdasR K78Q , and OdasR K78R strains were cultivated onReversion 2 agar containing 0.5% yeast extract (R2YE) (36) at 30°C.The developmental phenotypes were examined by scanning electron microscopy (SEM), as shown in Fig. 5.The strains overexpressing DisA K66Q showed a vegetative growth phenotype.In contrast, when DisA K66R was overexpressed, we observed dramatic hypersporulation (Fig. 5), revealing that elevated c-di-AMP accumulation contributes to sporulation in S. erythraea and that K66 acetylation abolishes this facilitation.Compared with OdasR K78R , OdasR K78Q showed delayed differentiation and sporulation (Fig. 5).These data indicated that c-di-AMP levels in response to acetylation status are critical to developmental fate.

The physiological connection between AcP and c-di-AMP homeostasis is conserved
The fact that DisA_N domain-containing proteins (Pfam PF02457) are present in more than 11,000 bacterial and archaeal species raised the possibility that a similar regulation by AcP-induced acetylation might broadly exist.To complement these evolutionary insights, DisA homologs from Streptomyces lividans, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces venezuelae, Streptosporangium roseum, and Thermomonospora curvata were screened as representative actinobacteria for phyloge netic surveys.Indeed, according to the alignment of the S. erythraea DisA sequence with its homologs, K66 was highly conserved (Fig. 6A; Fig. S10).Intriguingly, we found the same results for DasR (Fig. 6B; Fig. S11).We predict, therefore, that a similar depend ence on AcP contributes to c-di-AMP homeostasis and that changes in morphological differentiation may occur within actinobacteria subjected to constant nutrient stress.

DISCUSSION
c-di-AMP governs numerous essential processes, including the osmotic state, biofilm formation, acid stress resistance, and the response to DNA damage and other functions, which have been studied extensively in Gram-negative bacteria (2).Variations in the c-di-AMP level (both high and low c-di-AMP levels) cause metabolic imbalance, which alters cell proliferation and related metabolic pathways (18).Mechanisms that ensure c-di-AMP maintenance are therefore crucial for cell function and have been the focus of intense scrutiny.Here, we identified a noncanonical route for DisA and hence c-di-AMP, which is modulated by direct or indirect regulation of AcP-dependent acetylation.Generally, DisA-like proteins are the most common group of DAC domain-contain ing proteins in which the DAC domain is connected through a specific linker region to a DNA-binding domain.Such DisA proteins are mostly present in Gram-positive spore-forming Bacillus and Actinobacteria.DisA forms a stable octamer with central pairs of DAC domains in solution, and the structure of the DisA octamer offers a plausible mechanism for the regulation of DAC activity (19).Indeed, our results indicated that DisA acetylation at K66 strongly inhibits diadenylate cyclase with a reduction in octamer formation.Such a conformational change in the protein could be the basis for the lower capability of four-way substrate binding.The intracellular c-di-AMP levels measured from cell extracts of S. erythraea WT strains under N + or N − conditions showed a decrease in the c-di-AMP concentration under N − conditions compared to that under N + conditions (Fig. S12), demonstrating that global hyperacetylation inhibited c-di-AMP accumulation.
In addition to the posttranscriptional regulation of its enzymatic activity, the transcription of disA is under the direct transcriptional repression of the global plei otropic regulator DasR, which is also regulated by AcP-related acetylation, to some extent resembling allosteric regulation.The network between AcP and DisA forms a homeostatic signal loop to maintain appropriate c-di-AMP states within the bacterial cell (Fig. 7), exhibiting therapeutic or industrial potential for preventing microbial growth.Intracellular survival within macrophages, which are thought to provide a nutritionally restricted environment, is important for mycobacterial pathogenesis.Although the mechanisms by which M. tuberculosis persists in macrophages remain largely unknown, intermediate metabolites, including AcP, acetyl-CoA, propionyl-CoA, and succinyl-CoA, are good targets for understanding the pathogenesis of mycobacteria.Importantly, it was suggested that, in addition to the primary metabolic defects, this metaboliteinduced acylation modification could alter protein function and thus contribute to the pathophysiology.Notably, we found that AcP-dependent acetylation of DasR led to a loss of function in dimerization, which caused weakened DasR-DNA binding and inhibited its transcriptional activity, possibly exerting a global impact on actinobacterial physiology.
AcP-induced protein acetylation is generally considered a consequence of carbon overflow or an environmental carbon-nitrogen imbalance (24,37,38).Therefore, environmental cues can be integrated into a signaling network by affecting the activity of DisA, thereby controlling downstream phenotypic output.In all domains of life, nucleotide-based second messengers allow rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions.Previous studies have well explored that c-di-GMP acts as a brake on developmental transition through BldD and WhiG in the Streptomyces life cycle (20,34).Based on our results, c-di-AMP has the opposite effect on developmental transition, which is under the fine-tuned regulation of AcP.It probably offers diverse and cooperative functions among different second messengers to recruit an appropriate signaling molecule and to decide between growth and sporulation.Due to the high conservation of the identified crucial sites (K66 of DisA and K78 of DasR) and the widespread AcP-triggered acetyla tion in bacteria, it is intriguing to speculate that the regulatory link between c-di-AMP homeostasis and the AcP signal in response to environmental stimuli is conserved in bacteria even though the specific molecular mechanisms may be completely different.
In summary, the identification of AcP-dependent acetylation established a network for DisA regulation at both the transcriptional and posttranslational levels, which contributes to global c-di-AMP homeostasis.Fundamental studies of c-di-AMP signaling have also revealed significant therapeutic promise.The decreased virulence associated with elevated c-di-AMP suggests that regulation of its production or hydrolysis may hold promise for therapeutic intervention in bacterial infections.Furthermore, the potent immunostimulatory activity of c-di-AMP and other cyclic dinucleotides shows promise as vaccine adjuvants for the prevention of infectious and malignant diseases.It also predicts that the regulatory network and the key acetylation sites described here may, in some instances, be a concept with important consequences for pathogen biology and new therapeutic discovery efforts.

Bacterial strains and culture conditions
All strains and plasmids used in this study are listed in Table S3.The activation and preservation of all strains were described in detail in previous studies (39,40).The S. erythraea wild-type strain (NRRL2338) and mutants were cultured in trytone soy broth or minimal Evans medium {25-mM N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid sodium salt, 10-mM KCl, 2-mM Na 2 SO 4 , 2-mM citric acid, 0.25-mM CaCl

Overproduction and purification of proteins
The primers used for the amplification of disA and dasR from S. erythraea genomic DNA are listed in Table S4.The details of protein expression and purification were described in our previous works (39,40).The purified proteins were analyzed by SDS-PAGE and the protein concentration was determined using BCA Protein Assay Kit [Tiangen Biotech (Beijing) Co., Ltd] with bovine serum albumin (BSA) as the standard.

In vitro protein acetylation assays
In vitro acetylation assays with AcP were performed as previously described (41).Next, 10 µg of purified protein was added to a reaction mixture (total of 100 µL) containing 50-mM HEPES (pH 7.5) and 10-mM AcP.To investigate the effect of acetylation on the function of DisA/DasR, DisA/DasR and its mutants were incubated with or without AcP for 5 h.DisA WT /DasR WT incubated without AcP for 5 h was used as a control, which could exclude the effects caused by the incubation periods.After incubation, the reaction samples were analyzed by Western blotting and LC-MS/MS.

Identification of acetylated residues by LC-MS/MS
Protein samples were first separated by SDS-PAGE.The bands were then destained and dehydrated for further digestion and LC-MS/MS as described in detail in previous studies (42).The gel bands were sliced and destained in 50% ethanol.After being fully dehydra ted in 100% acetonitrile (ACN), the samples were reduced by 10-mM dithiothreitol at 56°C for 40 min and then alkylated by 15-mM iodoacetamide in darkness for another 40 min.Then, the gels were washed in washing buffer [50% ACN/50% 50-mM NH 4 HCO 3 (vol/vol)], and the proteins were digested by trypsin at an enzyme-to-substrate ratio of 1:40 for 16 h.The tryptic peptides were extracted in 50% ACN/5% trifluoroacetic acid (TFA), 75%/0.1% TFA, and 100% TFA in sequence.The samples were then dissolved in solvent A [0.1% (vol/vol) formic acid and 2% acetonitrile in water] and analyzed by an Orbitrap Fusion mass spectrometer in two technical replicates.The raw data were converted to mgf files and then analyzed by the Mascot search engine (v.2.3.01,Matrix Science).The search parameters were as follows: enzymes, trypsin/P; missed cleavage, 2; fixed modification, carbamidomethyl (C); variable modifications, acetylation (K), oxidation (M), and acetyl (protein N-terminal); peptide mass tolerance, 10 ppm; and fragment mass tolerance, 0.5 Da.The areas under the curve of the precursor ion peak were used to evaluate the peptide intensity.The ratios of the acylated peptides were normalized to the corresponding protein levels.Normalized ratios of the peptides were used for further analysis.

Site-directed mutagenesis of acetylated site mutants
Using the primers listed in Table S4 and a fast mutagenesis system (TransGen Biotech, China), we introduced the mutants (K78Q and K78R) of DasR and the mutants (K66Q and K66R) of DisA into the pET28a (+) plasmid.After DNA sequencing confirmation, the recombinant plasmids were introduced into E. coli BL21 (DE3).The selected mutants were verified by PCR and DNA sequencing.

IP and IB
Purification of the DisA and DasR proteins from S. erythraea through IP was performed as previously described (40) using specific anti-DisA and anti-DasR polyclonal antibodies (Solarbio, China), and the acetylation level was tested by IB using an anti-AcK antibody (PTM-102, HangZhou Jingjie).The binding signal was visualized using an Omni-ECL Enhanced Pico Light Chemiluminescence Kit (Shanghai Epizyme Biomedical Technology Co., Ltd.) and scanned with a Tanon 4600 (Biotanon, China).

Overexpression of DisA/DasR and its mutants in S. erythraea
The indicated strains were constructed as previously described (24).Briefly, overexpres sion plasmids were constructed by inserting the indicated sequence into the integrative shuttle vector pIB139 (33) between the NdeI and EcoRV restriction sites.This vector has been verified to have no effect on the bacterial physiology (43).The primers used are listed in Table S4.The recombinant plasmids were transformed into S. erythraea competent cells via polyethylene glycol and then integrated into the genome via phiC31.Apramycin resistance was used for selection.The selected mutants were confirmed by PCR and DNA sequencing.

c-di-AMP quantification
As described previously by Corrigan et al. (44) with some modifications.Bacteria were collected by centrifugation (10,000 rpm, 30 min), washed, and freeze-dried after centrifugation to determine the dry weight for standardization purposes.The precipi tate was suspended in 15-20 mL of ice-cold cell extraction buffer (ether:methanol:H 2 O, 40:40:20; liquid chromatography grade, VWR(Shanghai) Co.,Ltd, rapid freezing for 5 min).The samples were instantly frozen in liquid N 2 for 10 min, heated to 95°C for 10 min, and held at 4°C for 30 min.Then, a cell crushing machine was used for ultrasonic lysis for 15 min.The supernatant was removed and stored at 4°C.The cell components were mixed and washed with extraction buffer, incubated on ice for 30 min and heated again.The sample was centrifuged again, and the supernatant was mixed with the previous supernatant.After freeze-drying, the samples were resuspended in 500 µL of cell extraction buffer for high-performance liquid chromatography (HPLC) (45) with a chromatographic analysis column RPC-18 (250 × 4.5 mm, Kromasil).

DAC assays
DAC assays were performed as previously described (46).The reaction mixtures with purified 10-µM DisA protein (monomer concentration) in 0.1-M NaCl, 40-mM Tris (pH 7.5), and 10-mM MgCl 2 were started by the addition of 100-mM ATP.At regular time intervals (every 15 min), the reactions were stopped with an equal volume of 0.5-M EDTA (pH 8.0).The samples were then analyzed for c-di-AMP production by HPLC.Enzyme activity was expressed as specific activity where unit of activity is the change in c-di-AMP production per minute per milligram of protein.

EMSA
EMSAs were performed as previously described (30).DNA fragments and the upstream region from −300 to +50 of the detected genes were amplified by PCR using the primers listed in Table S4.The initial product was conjugated to 5′-biotin-AGCCAGT GGCGATAAG-3′ and purified with a HiPure PCR Pure Mini Kit (Magen, China).EMSAs were performed with a chemiluminescent EMSA kit (Beyotime Biotechnology, China) in a 10-µL reaction mixture containing 5-nM DNA and purified DasR.The DNA-protein mixture was incubated at 18°C for 30 min.Afterward, the samples were separated on a 6.5% polyacrylamide gel (Acr-Bis, 30%, 29:1) in 0.5 × Tris-borate-EDTA buffer at 380 mA.The bands were detected by an Omni-ECL Enhanced Pico Light Chemiluminescence Kit (Shanghai Epizyme Biomedical Technology Co., Ltd.) with a Tanon 4600 system (Biotanon).

CD spectroscopy
CD spectroscopy was performed as previously described (47).The purified protein was replaced with CD buffer (1.4-M KF, 100-mM K 2 HPO 4 , and 18-mM KH 2 PO 4 , pH 7.4) and fixed to 0.2 mg/mL.Samples were measured at wavelengths of 180-260 nm at 25°C.For the analysis of the data, the background buffer signal was subtracted.After the processed data were exported, CDNN CD spectra deconvolution software (Applied Photophysics) was used for analysis of the secondary structure of the protein.

BLI assay
A streptavidin (SA) biosensor purchased from ForteBio was used in this work.The loading buffer (pH 8.0) contained 10-mM HEPES, 2-mM MgCl 2 , 0.1-mM EDTA, and 200-mM KCl, and the running buffer contained an extra 10-µg/mL BSA and 0.02% Tween-20.The biotin-labeled DNA probe used was the same as that used for the EMSAs.The DNA probe was stored in loading buffer, and His-tagged DasR was stored in running buffer during the BLI assay with SA sensors.Samples were then detected within the OptiPlate-96 Black Opaque (PerkinElmer).

ChIP-seq and data analysis
The DasR WT and DasR K78Q strains were subsequently grown for the appropriate length of time.Formaldehyde was added to the cultures at a final concentration of 1% (vol/ vol), and the incubation was continued for 30 min.Glycine was then added to a final concentration of 125 mM to stop the cross-linking.The samples were left at room temperature for 10 min and washed twice in precooled TBS buffer (pH 7.5) containing 20-mM Tris-HCl and 150-mM NaCl.ChIP-seq was performed by E-GENE Tech Co., Ltd.(Shenzhen) using an anti-DasR polyclonal antibody.Briefly, fastp software (v.0.20.0) was used to trim adaptors and remove low-quality reads to obtain high-quality clean reads.Clean reads were aligned to the reference genome using Bowtie2 software (v.2.2.4).MACS2 software (v.2.2.7.1) was used for peak calling.Bedtools software (v.2.30.0) was used for peak annotation based on GTF annotation files.Homer (v.4.11) software was used to identify motifs.MAnorm2 (v.1.2.0) software was used to identify differentially enriched regions.The enriched peaks were visualized with Integrative Genomics Viewer (v.2.4.10) software.

Western blotting
Western blotting was performed as previously described (40).Protein lysates were separated by 10% SDS-PAGE and then transferred to PVDF membranes for 60 min at 100 V.After blocking with 3% BSA in phosphate-buffered saline containing 0.1% Tween-80 buffer at room temperature for 1 h, an anti-AcK antibody (PTM-102, HangZhou Jingjie) diluted 1:15,000 in Tris-buffered saline with tween (TBST)/5% BSA was used.The blot was performed via an ImageQuant LAS 4000 system (GE Healthcare, UK) after chemilumines cent Horseradish Peroxidase (HRP) substrate treatment.

RNA preparation and real-time RT-PCR
Total RNA extraction was performed according to our previous work (48).Total RNA (1 µg) was used to synthesize cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara).16S rRNA (SACE_8101) was used as the internal standard.The resulting cDNA was diluted and used as a template for real-time RT-PCR with SYBR Premix Ex Taq GC (Takara).The primers used are listed in Table S4.PCR assays were performed on a CFX96 real-time system (Bio-Rad, Hercules, CA) with the following PCR conditions: 95°C for 5 min, 40 cycles at 95°C for 5 s and at 60°C-64°C for 30 s, and an extension at 72°C for 10 min.The transcriptional variations were analyzed by the threshold cycle (2 −∆∆CT ) method.

Scanning electron microscopy
S. erythraea strains were cultivated on R2YE agar plates (36) covered with plastic cellophane at 30°C.At the specified times, a piece of cellophane covered with mycelia was extracted, fixed with 2% osmium tetroxide for 24 h, and then dehydrated by air drying for 1 h.Each specimen was sputter coated with platinum gold and examined with a Gemini SEM 500 scanning electron microscope.

FIG 1
FIG 1 AcP-induced acetylation of DisA impairs its DAC activity.(A) Acetylation level of DisA from the S. erythraea wild type (WT) strain under excess nitrogen (N + ) or limited nitrogen (N − ) conditions.Each lane was loaded with an equal amount of DisA protein.(B) In vitro acetylation of His-tagged DisA protein with 10-mM AcP for various lengths of time (0, 1, 3, and 5 h) at 37°C.Acetylation levels were determined by Western blotting with a specific anti-AcK antibody.(C) DAC activity of the DisA WT and DisA AcP proteins.The DAC activity of DisA WT was set to 100%.(D) CD spectra of the DisA WT and DisA AcP proteins.(E) Domain organization of the DisA is shown in differently colored boxes.The red inverted triangle shows the position of K66.(F) Cross-linking of the DisA WT , DisA AcP , DisA K66Q , and DisA K66R proteins.The band intensities were quantified by densitometry using ImageJ software.The relative densitometry of the monomer and octamer in the DisA was set to 100.(G) DAC activity of the DisA WT , DisA K66Q , and DisA K66R proteins.The DAC activity of DisA WT was set to 100%.The error bars show the SDs of three independent experiments.Ordinary one-way analysis of variance was used for the statistical test.

FIG 2
FIG 2 Effect of K66 acetylation on the synthesis of c-di-AMP in vivo.(A) Growth curves of the S. erythraea WT, OdisA WT , OdisA K66Q , and OdisA K66R strains grown at 30°C in trytone soy broth (TSB) media.(B) The disA transcription levels in the WT, OdisA WT , OdisA K66Q , and OdisA K66R strains grown in TSB media.The fold change represents the expression level compared to that of disA in the WT strain.(C) Intracellular c-di-AMP levels in cell extracts of the S. erythraea WT, OdisA WT , OdisA K66Q , and OdisA K66R strains grown in TSB media.The c-di-AMP concentrations of the samples were normalized to the dry cell weight.The error bars show the SDs of three independent experiments.Ordinary one-way analysis of variance was used for the statistical test.

FIG 3
FIG 3 AcP-induced acetylation of DasR blocks its DNA-binding activity.(A) Acetylation level of DasR in the S. erythraea WT strain under N + or N − conditions.Each lane was loaded with an equal amount of DasR protein.(B) In vitro acetylation of His-tagged DasR protein with 10 mM AcP for various lengths of time (0, 1, 3, and 5 h) at 37°C.Acetylation levels were determined by Western blotting using a specific anti-AcK antibody.(C) EMSA of DasR WT and DasR AcP binding to its target gene disA promoter.(D) CD spectra of the DasR WT and DasR AcP proteins.(E) The domain organization of DasR is shown in differently colored boxes.The red inverted triangle shows the position of K78.(F) EMSA of DasR and its mutants binding to its target gene disA promoter.(G) Biolayer interferometry assay of purified His-DasR, DasR AcP , DasR K78Q , and DasR K78R with the disA gene promoter.(H) Cross-linking of DasR and its mutants.The band intensities were quantified by densitometry using ImageJ software.The relative densitometry of the monomer and dimer in DasR WT was set to 100.

FIG 4
FIG 4 K78 acetylation releases the DasR-mediated repression of disA in vivo.(A) Heatmaps of the ChIP-seq signal density at the peak center and transcription start sites (±2 kb).The average signal profile is shown.Red indicates a low signal, and blue indicates a high signal.Source data are provided as a source data file.(B) ChIP-seq signal in the indicated strains.(C) Integrative Genomics Viewer tracks showing ChIP-seq signals at the promoter regions of disA in the indicated strains.(D) The transcription levels of disA in the indicated strains.The fold change represents the expression level compared to that of the DasR WT strain.(E) Intracellular c-di-AMP levels in the indicated strains.The c-di-AMP concentrations of the samples were normalized to the dry cell weight.The error bars show the SDs of three independent experiments.A t-test was used for the statistical analysis.

FIG 5
FIG 5 AcP-induced acetylation influences sporulation.Scanning electron micrographs of the S. erythraea WT, OdisA K66Q , OdisA K66R , OdasR K78Q , and OdasR K78R strains grown for 96 h on R2YE agar at 30°C.The images are shown at ×5,000 magnification.Representative pictures of two independent experiments with similar results are shown.

FIG 6
FIG 6 Sequence alignment of DasR and DisA proteins within actinobacteria.Alignments of the sequences of the Streptomyces lividans, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces venezuelae, Streptosporangium roseum, Thermomonospora curvata, and S. erythraea DisA (A) and DasR (B) proteins.The conserved lysine of all species is shown in bold red.

FIG 7
FIG 7 Schematic representation of the pathways involved in AcP control of c-di-AMP homeostasis.In the presence of AcP, the DasR residue K78 and the DisA residue K66 are acetylated (in red).DasR acetylation at K78 weakened its DNA binding and inhibited its transcriptional activity.Since DasR functions predominantly as a global repressor and has a negative effect on disA expression, the reduced DasR-DNA interaction caused by acetylation reversed the repression of disA and resulted in elevated disA transcription.DisA acetylation at K66 prevented its DAC activity, which inhibited c-di-AMP synthesis.PTM, posttranslational modification.